Alzheimer's disease (AD) is a progressive neurodegenerative disorder that destroys higher brain structures, such as those involved in memory and cognition. The disease leads to deficits in cognitive function and declines in memory, learning, language, and in the ability to perform intentional and purposeful movements. There is a need for effective methods and compositions for treatment and prophylaxis of AD.
AD is histologically characterized by the presence of extraneuronal plaques and intracellular and extracellular neurofibrillary tangles in the brain. Plaques are composed mainly of β amyloid (Aβ), whereas tangles comprise pathological forms of tau, such as pathological tau conformers and their aggregates. A recognized role for tau in AD pathology has been demonstrated in numerous studies. For example, Braak showed that the closest correlate for AD neurodegeneration was the presence of tau tangles, and not of amyloid plaques (Braak, H., et al. Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol 82:239-259 (1991)).
Tau belongs to a family of intrinsically disordered proteins, characterized by the absence of a rigid three-dimensional structure in their physiological environment (Skrabana et al., 2006). However, tau truncation and hyperphosphorylation can cause pathological transformations from an intrinsically disordered state to multiple soluble and insoluble misdisordered structures, including paired helical filaments (PHFs) and other aggregates (Wischik, C. M., Novak, M., Edwards, P. C., Klug, A., Tichelaar, W., Crowther, R. A. (1988). Structural characterization of the core of the paired helical filament of Alzheimer disease, Proc Natl Acad Sci USA 85, 4884-8; Wischik, C. M., Novak, M. Thøgersen, H. C., Edwards, P. C., Runswick, M. J., Jakes, R., Walker, J. E., Milstein, C., Roth, M., Klug, A. (1988). Isolation of a fragment of tau derived from the core of the paired helical filament of Alzheimer disease, Proc Natl Acad Sci USA 85, 4506-10; Novak et al., 1993; Skrabana et al., 2006; Zilka, N., et al. Chaperone-like Antibodies Targeting Misfolded Tau Protein: New Vistas in the Immunotherapy of Neurodegenerative Foldopathies. Journal of Alzheimer's disease 15 (2008) 169-179; Kovacech B, Novak M. (2010). Tau truncation is a productive posttranslational modification of neurofibrillary degeneration in Alzheimer's disease. Curr Alzheimer Res December; 7(8):708-16); Kovacech B, Skrabana R, Novak M. (2010). Transition of tau protein from disordered to misordered in Alzheimer's disease. Neurodegener Dis 7: 24-27). These structural changes lead to a toxic gain of function, to a loss of physiological function of the native protein, or both (Zilka et al., 2008; Kovacech B, Novak M. (2010). Tau truncation is a productive posttranslational modification of neurofibrillary degeneration in Alzheimer's disease. Curr Alzheimer Res December; 7(8):708-16); Kovacech B, Skrabana R, Novak M. (2010). Transition of tau protein from disordered to misordered in Alzheimer's disease. Neurodegener Dis 7: 24-27).
Tau's physiological function is in mediating the assembly of tubulin monomers into microtubules that constitute the neuronal microtubules network (Buee, L., Bussiere, T., Buee-Scherrer, V., Delacourte, A., Hof, P. R. (2000). Tau protein isoforms, phosphorylation and role in neurodegenerative disorders. Brain Research. Brain Research Reviews. 33, 95-130). Tau binds to microtubules through repetitive regions located in the C-terminal portion of the protein. Butner K A, Kirschner M W. 1991. Tau protein binds to microtubules through a flexible array of distributed weak sites. J Cell Biol 115: 717-730; Lee G, Neve R L, Kosik K S. 1989. The microtubule binding domain of tau protein. Neuron 2: 1615-1624. These repeat domains (R1-R4), are not identical to each other, but comprise highly conserved 31-32 amino acids (Taniguchi T, Sumida M, Hiraoka S, Tomoo K, Kakehi T, Minoura K, Sugiyama S, Inaka K, Ishida T, Saito N, Tanaka C 2005 (Effects of different anti-tau antibodies on tau fibrillogenesis: RTA-1 and RTA-2 counteract tau aggregation. FEBS Lett 579:1399-1404; Taniguchi S, Suzuki N, Masuda M, Hisanaga S, Iwatsubo T, Goedert M, Hasegawa M. Inhibition of heparin-induced tau filament formation by phenothiazines, polyphenols, and porphyrins. J Biol Chem 280:7614-7623 (2005)). In the human brain, there are six unique isoforms of tau, which differ from each other in the presence or absence of certain amino acids in the N-terminal portion of tau, in combination with either three (R1, R3, and R4) or four (R1-R4) repeat domains, at the C-terminal end of the protein. See also FIG. 1, which shows the six human isoforms (2N4R, 1N4R, 2N3R, 0N4R, 1N3R, and 0N3R SEQ ID Nos. 151-156, respectively, in order of appearance).). It has been proposed that the most potent part of tau to induce microtubule polymerization are the sequences 306-VQIVYK-311 (SEQ ID NO: 146) and 274-KVQIINKK-281 region (SEQ ID NO: 144), overlapping R1-R2. (von Bergen M, Friedhoff P, Biernat J, Heberle J, Mandelkow E M, Mandelkow E. 2000. Assembly of tau protein into Alzheimer paired helical filaments depends on a local sequence motif ((306)VQIVYK(311)) forming beta structure. Proc Natl Acad Sci USA 97: 5129-5134.) Id.
In addition, tau's pathological and physiological functions appear to be influenced by the specific structural conformation, and the intrinsically disordered structure, adopted by the full length protein isoforms and their fragments. For example, Kontsekova et al. described a conformational region (encompassing residues 297-IKHVPGGGSVQIVYKPVDLSKVTSKCGSL-325 (SEQ ID NO: 145) within certain truncated tau molecules which had a significant relationship to the function of those truncated tau molecules on microtubule assembly (WO 2004/007547).
In addition to their physiological role, tau repeats are believed to participate in the formation of pathological tau aggregates and other structures. Thus, there is a need for tau-targeted therapeutic and diagnostic approaches that are capable of discriminating between physiological and pathological microtubule binding repeat region-mediated activities. For example, the pronase resistant core of pathological paired helical filaments (PHFs) consists of the microtubule binding regions of 3- and 4-repeat tau isoforms (Jakes, R., Novak, M., Davison, M., Wischik, C. M. (1991)). Identification of 3- and 4-repeat tau isoforms within the PHF in Alzheimer's disease. EMBO J 10, 2725-2729; Wischik, et al. 1988a; Wischik, et al. 1988b). Further, Novak et al. showed that the protease resistant core of the PHFs, which is 93-95 amino acids long, was restricted to three tandem repeats (Novak, M., Kabat, J., Wischik, C. M. (1993). Molecular characterization of the minimal protease resistant tau unit of the Alzheimer's disease paired helical filament. EMBO J 12, 365-70). Von Bergen et al. determined a minimal-tau peptide/interaction motif (306-VQIVYK-311, SEQ ID NO: 146), as well as a second site on tau (275-VQIINK-280) (SEQ ID NO: 147), which form beta-sheets and are described as potentially responsible for initiating the formation of PHFs, a pathological tau aggregate (von Bergen M, Friedhoff P, Biernat J, Heberle J, Mandelkow E M, Mandelkow E. 2000. Assembly of tau protein into Alzheimer paired helical filaments depends on a local sequence motif ((306)VQIVYK(311)) forming beta structure. Proc Natl Acad Sci USA 97: 5129-5134); EP 1214598; WO 2001/18546). See FIG. 2 for a functional map of tau. Consequently, current strategies aim at generating anti-aggregating drugs that do not disrupt tau's intracellular role in microtubule stabilization.
Moreover, while under physiological circumstances tau is considered an intracellular cytoplasmic protein, intracellular tau can be released into the extracellular space and contribute to neurodegeneration (Gómez-Ramos, A., Diaz-Hernández, M., Cuadros, R., Hernández, F., and Avila, J. (2006). Extracellular tau is toxic to neuronal cells. FEBS Lett 580(20), 4842-50). Indeed, neuronal loss has been linked to the topographic distribution of neurofibrillary tangles (made up of tau protein) in AD brains (West, M. J., Coleman, P. D., Flood, D. G., Troncoso, J. C. (1994). Differences in the pattern of hippocampal neuronal loss in normal ageing and Alzheimer's disease. Lancet 344, 769-72; Gómez-Isla, T., Price, J. L., McKeel Jr, D. W., Morris, J. C., Growdon, J. H., Hyman, B. T. (1996). Profound loss of layer II entorhinal cortex neurons occurs in very mild Alzheimer's disease. J Neurosci 16(14), 4491-500, Gomez-Isla T, Hollister R, West H, Mui S, Growdon J H, Petersen R C, Parisi J E, Hyman B T. Neuronal loss correlates with but exceeds neurofibrillary tangles in Alzheimer's disease. Ann Neurol 41:17-24 (1997)). Further, the levels of total tau and phosphorylated tau are increased in the cerebrospinal fluid (CSF) of patients with AD (Hampel, H., Blennow, K., Shaw, L. M., Hoessler, Y. C., Zetterberg, H., Trojanowski, J. Q. (2010). Total and phosphorylated tau protein as biological markers of Alzheimer's disease. Exp Gerontol 45(1), 30-40), and extracellular tau has been described as “ghost tangles” in the brain (Frost, B., Diamond, M. I. (2009). The expanding realm of prion phenomena in neurodegenerative disease. Prion 3(2):74-7), indicating that intracellular tau is released into extracellular space. In addition, extracellular tau aggregates can enter cells and stimulate fibrillization of intracellular tau, further seeding tau monomer for production of pathological tau aggregates (Frost et al., 2009). Such studies have highlighted that extracellular, insoluble tau could act as a transmissible agent to spread tau pathology throughout the brain in a prion-like fashion (Frost, B., Jacks, R. L., Diamond, M. I. (2009). Propagation of tau misfolding from the outside to the inside of a cell. J Biol Chem 284(19), 12845-52, Frost et al., 2009; Frost, B., Diamond, M. I. (2009). The expanding realm of prion phenomena in neurodegenerative disease. Prion 3(2):74-7). Targeting abnormal tau can reduce tau-associated extracellular and intracellular pathology. See, Eva Kontsekova, Norbert Zilka, Branislav Kovacech, Petr Novak, Michal Novak. 2014. First-in-man tau vaccine targeting structural determinants essential for pathological tau-tau interaction reduces tau oligomerisation and neurofibrillary degeneration in an Alzheimer's disease model. Alzheimer's Research & Therapy, 6:44. Therefore, there is a need for treatments capable of decreasing extracellular tau, either by impeding its formation, promoting its clearance, or both, as well as for treatments that decrease intracellular disease tau. An increased understanding of the molecular mechanisms underlying the pathological transformations of tau has opened up the possibility of specifically targeting pathological modifications of tau for therapeutic purposes.
International Publication No. WO2013/041962 by Novak et al. describes the discovery of four regions of tau that promote tau-tau aggregation in AD and antibodies that prevent tau aggregation by binding to those four regions.
Although other studies have described antibodies that bind to tau sequences, and some of those antibodies also reportedly interfere with tau aggregation and clearance (Asuni A A, Boutajangout A, Quartermain D, Sigurdsson E M. Immunotherapy targeting pathological tau conformers in a tangle mouse model reduces brain pathology with associated functional improvements. J Neurosci 27:9115-9129 (2007)), no monoclonal anti-tau antibody is yet reportedly undergoing clinical trials in AD.
The success of foreign (mouse) monoclonal antibodies in human treatment has been, in part, impeded by immunogenic antiglobulin responses mounted by the human recipient against such foreign therapeutics. These complicate both the safety and pharmacokinetic properties of antibodies. These challenges have led to the development of engineered antibodies that carry a lower risk of immune reactions. A variety of patented engineering technologies (e.g., chimerization, humanization, CDR grafting, framework grafting, affinity maturation, phage display, transgenic mice) are constantly being developed to facilitate this process. For recent review, see Safdari Y1, Farajnia S, Asgharzadeh M, Khalili M. Antibody humanization methods—a review and update. 2013. Biotechnol Genet Eng Rev. 29:175-86. doi:10.1080/02648725.2013.801235 and Almagro JC1, Fransson J. Humanization of antibodies. 2008. Front Biosci. 13:1619-33.
Humanized antibodies are designed, primarily, to retain the specificity and affinity of the parent antibody while having human constant regions, which ideally would present less of an immunogenic target to the patient. The typical humanized antibody carries the complementarity determining regions (CDRs) of a parent antibody of mouse or rat origin, and framework regions (FR) and constant regions that are mostly of human origin but have often been mutated to retain the parent antibody's binding properties. But antigen-binding affinity and specificity are not the only factors affecting the biological activity and clinical success of an antibody. Improving an antibody's activation of the patient's immune system is key to the value of some humanized antibodies, whereas for others reduction of cellular-mediated toxicity is a goal. Ultimately, an increased understanding of antibody structure and activity allows researchers to engineer, often through mutations, more advanced humanized antibodies that are more homogeneous with better antigen binding properties (binding affinity, target specificity), effector functions, stability, expression level, purification properties, pharmacokinetics, and pharmacodynamics. Many of these improvements are important for the commercial viability of a given antibody. Sometimes, after target binding affinity and specificity is achieved, it is necessary to mutate some of the amino acids in the CDRs or FRs to decrease a humanized antibody's susceptibility to aggregation. Other times, the constant regions are altered (switched or mutated) for improved effector functions. These and other aspects of antibody function and activity continue to present challenges to the development of antibodies for clinical use. Described below is a set of humanized antibodies against tau that have been engineered to possess unexpected advantageous properties. Also provided below are novel methods and compositions comprising these highly specific and highly effective antibodies having the ability to specifically recognize and bind to pathological tau, impeding its aggregation. All these antibodies, methods, and compositions are useful for diagnosis and treatment of AD and related tauopathies.